Willams e medium

Hepatocytes were isolated from control and L-G6pc-/- mice at 12 WP using a two-step collagenase perfusion method. Liver was perfused via the portal vein with liver perfusion medium (Gibco, Waltham, MA) for 5 min at 37°C, followed by liver digest medium (Gibco) for 5 min at 37°C. The excised liver was incubated in liver digest medium for 30 min at 37°C, and then passed through a 100 μm cell strainer (Falcon, Franklin Lakes, NJ). The hepatocytes were pelleted by centrifugation at 4°C, washed twice with hepatocyte wash medium (Gibco), and purified via 20% Percoll gradients (GE Healthcare, Waukesha, WI). The resulting hepatocytes were washed with Willams E medium (Gibco) and resuspended in HepatoZYME-SFM (Gibco).

All procedures involving animals were approved by Changshu Institute of Technology Institutional Animal Care and Use Committee and conformed to the Guide for the Care and Use of Laboratory Animals of Jiangsu Province. All efforts were made to minimize suffering. Primary chicken hepatocytes were isolated from 4 female, 4-week-old Arbor Acres commercial chickens which were fasted 12 hours (h) before being anaesthetized by intraperitoneal injection of sodium thiopenthal (50 mg/kg) and anticoagulated by intraperitoneal injection of heparin (1750 U/kg). Livers were isolated and the chickens were sacrificed by removal of the hearts. Hepatocytes were isolated from the livers as previously described [30] (link). Hepatocytes from individual chickens were cultured separately. Hepatocytes were plated in 24-well plates or 10-cm dishes at a density of 1.3×10⁶ cells/ml in Willam’s E medium (Gibco, Grand Island, NY) supplemented with 5% chicken serum, 100 U/ml penicillin-streptomycin, 10 µg/ml insulin and 30 mmol/L NaCl in a humidified incubator at 37°C with 5% CO₂. Twenty-eight h later, hepatocytes were serum starved for 8 h, followed by 12 h of treatment with 500 ng/ml chicken GH (chGH) (Prospec, Ness-Ziona, Israel) or an equal volume of PBS. Cells were lysed and total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s directions.